Molecular Mechanisms of Fructose-induced Colorectal Cancer Cell Survival

项目摘要 我们所吃的食物与疾病的发展和进展之间已经建立了明确的联系。 结直肠癌（CRC）。例如，果糖的消费增加了CRC发展的风险 和CRC特异性死亡率。然而，这种关联的机制尚不清楚。我们已经表明 每日适量口服高果糖玉米糖浆（HFCS，果糖和葡萄糖的混合物）会导致 更大和更具侵略性的肠腺瘤。这些效应在遗传性的小鼠中是不存在的。 己酮糖激酶（KHK）缺乏，该酶将果糖转化为1-磷酸果糖（F1 P）。一 这些肿瘤的代谢组学分析表明，在HFCS暴露后，F1 P是高度丰富的， 增加与丙酮酸激酶（PK）活性的降低相关。因此，我们假设F1 P， KHK的产物，通过作为PK的变构抑制剂促进合成代谢而增强肿瘤生长 代谢和细胞存活。我们将使用小鼠生理学和器官代谢，细胞 人类类器官培养和重组蛋白生物化学。在目标1中，我们将从基因和 在小鼠中操纵PK的M2同工酶（PKM 2）以询问其作为介导剂的作用。 HFCS诱导的肿瘤生长。在目标2中，我们将定义果糖暴露与 癌细胞存活率然而，我们发现，当暴露于果糖时，培养中的细胞不会生长得更快， 我们观察到细胞活力的显著改善，特别是在高细胞密度的条件下， 培养基中有果糖的缺氧。因此，我们推测F1 P通过抑制PKM 2，促进缺氧细胞凋亡， 生存我们将使用暴露于果糖和缺氧的细胞和类器官培养模型来测试这一假设。 我们将在这些模型中对KHK和PKM 2的表达和活性进行遗传学和生物学操作 以确定这些蛋白质对细胞代谢和存活的具体影响。在目标3中，我们将评估 F1 P对重组PK亚型的影响，特别关注PKM 2。我们假设果糖- 衍生的F1 P结合并抑制PKM 2。我们将进行生化活性和结构分析， 确定在F1 P存在下PK亚型的动力学参数和寡聚状态。这些 实验将揭示F1 P如何结合和抑制PKM 2的分子机制。总之，这些目标 将改变我们对果糖如何改变肿瘤细胞代谢的基本理解， 果糖/F1 P/PKM 2轴作为结直肠癌的代谢易损性，并为PKM 2 活化剂作为一种新的治疗方式，以打击CRC。

Project Summary Clear associations have been established between the food we eat and the development and progression of colorectal cancer (CRC). For example, the consumption of fructose increases the risk for CRC development and CRC-specific mortality. However, the mechanism underlying this association is unknown. We have shown that moderate daily exposure to oral high fructose corn syrup (HFCS, a mix of fructose and glucose) leads to larger and more aggressive intestinal adenomas in mice. These effects were absent in mice with genetic deficiency of ketohexokinase (KHK), the enzyme that converts fructose to fructose 1-phosphate (F1P). A metabolomic analysis of these tumors showed that F1P is highly abundant following HFCS exposure, and this increase correlates with a reduction in pyruvate kinase (PK) activity. Therefore, we hypothesize that F1P, the product of KHK, enhances tumor growth by acting as an allosteric inhibitor of PK to promote anabolic metabolism and cell survival. We will test this hypothesis using mouse physiology and organ metabolism, cell and human organoid culture, and recombinant protein biochemistry. In Aim 1, we will genetically and pharmacologically manipulate the M2 isozyme of PK (PKM2) in mice to interrogate its role as a mediator of HFCS-induced tumor growth. In Aim 2, we will define the mechanistic linkage between fructose exposure and cancer cell survival. We have found that cells in culture do not grow faster when exposed to fructose, however we observed a significant improvement in cell viability, especially under conditions of high cell density and hypoxia with fructose in the media. Therefore, we hypothesize that F1P inhibits PKM2 to promote hypoxic cell survival. We will test this hypothesis using cell and organoid culture models exposed to fructose and hypoxia. We will genetically and pharmacologically manipulate KHK and PKM2 expression and activity in these models to determine the specific effects of these proteins on cell metabolism and survival. In Aim 3, we will assess the effects of F1P on recombinant PK isoforms with a particular focus on PKM2. We hypothesize that fructose- derived F1P binds to and inhibits PKM2. We will perform biochemical activity and structural assays to determine the kinetic parameters and oligomeric state of PK isoforms in the presence of F1P. These experiments will reveal the molecular mechanisms of how F1P binds and inhibits PKM2. Together, these aims will change our fundamental understanding of how fructose alters tumor cell metabolism, define the fructose/F1P/PKM2 axis as a metabolic vulnerability of CRC, and provide pre-clinical evidence for PKM2 activators as a novel therapeutic modality to combat CRC.